The Influence of Hypoxia on Avoidance Learning, Intra-Cranial Self-Stimulation and Drinking Behavior

In this thesis we described the effects of hypoxia on three types of functional behavioL avoidance learning, self-stimulation and drinking behavior. Depth and induction method of hypoxia were chosen to secure maximal survival of the animals. The aim was to evaluate which behavioral paradigm could be used in pharmacological research for detection of drugs that could protect the brain against hypoxia. In the first paradigm (Chapter I) guinea-pigs were trained twice daily on five consecutive days to learn to avoid an electrical shock (unconditioned stimulus) upon presentation of an auditory stimulus (conditioned stimulus). In control animals the number of correct responses increased in the course of the training period. In addition, more correct responses were made during the second daily training session as compared to the first session. When the animals were exposed to hypoxia (1 min. 100% nitrogen) immediately after each training session acquisition was not attenuated. However, the level of performance during the second session was reduced to that seen in the first session of that same day. This effect was probably due to a blockade of either the development or retrieval of an intermediate memory trace which contained new information learned in the first session. Given both the stability and the specificity of the induced effect the paradigm seemed to be suitable for pharmacological research. Four compounds; flunarizine, nimodipine, nifedipine and verapamil were tested. Three of them were known to have anti-hypoxic effects in simple survival models. Only tlunarizine could antagonize the amnesic effects. Interestingly, it was also the only drug known to have direct effects on brain cells. In the next paradigm self-stimulation behavior was used (Chapter 3-6). Rats were trained to obtain electrical brain stimulation in the lateral hypothalamus by pressing a lever. Trained animals reached an average response rate of one hundred responses in one minute. This response rate was reduced immediately after exposure to normobaric hypoxia but it normalized within ten minutes. These effects were reduced after a second exposure to hypoxia. This form of adaptation did not continue after subsequent exposures. Self-stimulation was also significantly reduced when the sessions were held under hypobaric conditions. At a barometric pressure of 300 mm.Hg a significant reduction of the response rate was observed during the whole session. A progressive slow adaptation was seen after several sessions in the hypobaric environment

Quantitative definition and monitoring of the host cell protein proteome using iTRAQ: a study of an industrial mAb producing CHO-S cell line

There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs